PROJECT ABSTRACT Tumor infiltrating lymphocytes (TILs) can significantly increase the success of immunotherapy. Solid tumors present barriers to TIL penetration and function. Strategies to increase TILs and their function are challenged by the inability to direct the delivery of T cells into solid tumors and a lack of techniques to predict their uptake. Through this proposal, we will develop the use of image-guided irreversible electroporation (IRE) as a novel approach to promote TILs in solid tumors. IRE is clinically used for the treatment of solid tumors by applying microsecond long electric pulses that kill cells through rapid permeabilization of the cell membrane. A unique feature of IRE is that, while killing cells, it spares the extracellular matrix and blood vessels within the treatment region, which is ideal for stimulating local inflammation and promoting TILs. We will investigate our proposed strategy in translational mouse models and in patients with malignant pleural mesothelioma (MPM), which is a solid tumor where a higher ratio of cytotoxic TIL to immunosuppressor cells has already been shown to increase patient survival. In our experiments, we will use chimeric antigen receptor (CAR) T cells that are genetically modified to recognize mesothelin, a cell surface antigen that is overexpressed on mesothelioma cancer cells. Based on prior work by the PIs' team, we rationalize that cell death from IRE: (i) can be imaged using intra-procedural FDG-PET; (ii) can generate an intratumoral chemokine gradient that promotes TILs; and (iii) therefore, FDG-PET guided IRE can serve as a surrogate to both map and drive tumor-targeted CAR T-cell delivery. Furthermore, our strategy of IRE-directed CAR T-cell infiltration can be repeated using electric pulse parameters designed to be cancer cell-targeted/TIL-sparing, thereby augmenting immunotherapeutic efficacy. The long-term goal of our work is to develop image-guided IRE as a platform for predictable, reproducible, and controllable delivery of CAR T cells and other cellular anticancer therapies to solid tumors. In Aim 1, we will create an integrated tumor immune microenvironment map (iTIMM) combining FDG-PET imaging, computer simulations, and quantitative multiplex immunofluorescence histology to develop a tool that will determine T- cell localization in solid tumors treated with IRE. iTIMM will then be used to develop a clinically relevant protocol that, in combination of intravenous administration of T cells, will be used to increase TILs. In Aim 2, we will use dual imaging with PET (cancer cells) and bioluminescence imaging (T cells) to validate IRE pulse parameters to selectively enrich and increase functional CAR T cells within MPM. In Aim 3, we will perform a pilot clinical trial using PET-guided transthoracic IRE of MPM prior to resection to validate the concept of iTIMM and its effect on TILs. The modulation of the tumor microenvironment with image-guided IRE to map and localize T-cell delivery, promoting CAR T cell function, and translational validation of iTIMM presents innovative rational combinations of clinically used techniques and is an ambitious attempt to address a key challenge to solid tumor immunotherapy.